Unique CD8+ T Cell-Mediated Immune Responses Primed in the Liver

Abstract

Background: The liver immune environment is tightly regulated to balance immune activation with immune tolerance. Understanding the dominant immune pathways initiated in the liver is important because the liver is a site for cell transplantation, such as for islet and hepatocyte transplantation. The purpose of this study is to examine the consequences of alloimmune stimulation when allogeneic cells are transplanted to the liver in comparison to a different immune locale, such as the kidney.

Methods: We investigated cellular and humoral immune responses when allogeneic hepatocytes are transplanted directly to the recipient liver by intraportal injection. A heterotopic kidney engraftment site was used for comparison to immune activation in the liver microenvironment.

Results: Transplantation of allogeneic hepatocytes delivered directly to the liver, via recipient portal circulation, stimulated long-term, high magnitude CD8 T cell-mediated allocytotoxicity. CD8 T cells initiated significant in vivo allocytotoxicity as well as rapid rejection of hepatocytes transplanted to the liver even in the absence of secondary lymph nodes or CD4 T cells. In contrast, in the absence of recipient peripheral lymphoid tissue and CD4 T cells, CD8-mediated in vivo allocytotoxicity was abrogated, and rejection was delayed when hepatocellular allografts were transplanted to the kidney subcapsular site.

Conclusions: These results highlight the CD8-dominant proinflammatory immune responses unique to the liver microenvironment. Allogeneic cells transplanted directly to the liver do not enjoy immune privilege but rather require immunosuppression to prevent rejection by a robust and persistent CD8-dependent allocytotoxicity primed in the liver.

Figure 1. Allogeneic hepatocytes elicit rapid rejection whether transplanted directly (intraportal) or indirectly (intrasplenic) to the liver microenvironment

FVB/N (H-2^q) hepatocytes were transplanted into untreated wild-type (WT; H-2^b) recipients by intraportal injection (n=10) or by intrasplenic (n=15) injection. Graft rejection occurred promptly in both recipients transplanted directly to the liver by intraportal injection and those transplanted by conventional intrasplenic injection (MST=10 days, p=ns).

Figure 2. Alloimmunity initiated by hepatocellular transplantation directly (intraportal injection) to the liver induces high magnitude and persistent CD8-medated in vivo allocytotoxicity

FVB/N hepatocytes (H-2^q) were injected into untreated wild-type hosts (WT; H-2^b) by intraportal injection or by intrasplenic injection. A) Allospecific in vivo cytotoxic effector activity was measured serially following transplantation, in both recipient groups. Both groups developed high magnitude cytotoxicity by 7 days posttransplant (intraportal=87±4%, intrasplenic=90±2%), but intraportal injection uniquely resulted in sustained cytotoxic activity beyond the time of graft rejection (day 14=93±3%, day 21=90±4%, day 35=72±13%), whereas recipients receiving intrasplenic injection showed a sharp decrease in cytotoxicity following graft rejection (day 14=26±4%, day 21=16±6%, day 35=7±2%; p<0.01 for all time points, signified by “*”). Each data point represents n=4–6 animals per time point. B) CD8⁺ T cell depletion using monoclonal antibodies on days 6 and 7 posttransplant, 48 hours prior to the in vivo cytotoxicity assay (day 7 posttransplant), eliminated the cytotoxic effector function in hepatocyte recipients (6±2%; p<0.001, signified by “*”; n=4) demonstrating that in vivo effector function in hepatocyte recipients is CD8-mediated. The error bars represent standard error.

Figure 3. Rejection of allogeneic hepatocytes transplanted directly to the liver (intraportal injection) readily occurs in the absence of recipient peripheral lymphoid tissue

FVB/N hepatocytes (H-2^q) were transplanted through intraportal injection into untreated wild-type hosts (WT; n=10), WT hosts with previous splenectomy (splx; n=6), LTα KO hosts (lacking peripheral lymph nodes; n=6), and LTα KO hosts with previous splenectomy (n=6; all H-2^b). A) Graft rejection occurred with similar kinetics in all groups (MST=10 days; p=ns). CD8-depletion did not prolong graft survival in WT recipients (MST=day 14, n=4), whereas graft survival was significantly prolonged in CD8-depleted LTα KO recipients (MST>30; p<0.001; n=3). Prolonged graft survival was observed in WT recipients only following both CD4- and CD8-depletion (MST>30; p<0.001; n=4). B) Alloantibody production was evaluated in recipient mice on day 14 following transplantation. Following intraportal transplantation, WT recipients developed significant levels of alloantibody (titer=92±8; n=6) which was abrogated in CD4-deficient recipients (titer=12±2; n=5; p<0.001, signified by “*”) but remained intact despite splenectomy (splx; titer=75±14; n=4; p=ns). Significantly higher levels were observed following CD8-depletion in WT recipients (titer=1300±300, n=3, p<0.001 as compared to WT recipients, as signified by “**”). Alloantibody production was abrogated in WT recipients following CD4- and CD8-depletion (titer=8±2). Alloantibody was not detected in any lymph node deficient hosts (LTα KO; titer=13±3; n=4), LTα KO with splenectomy (splx; titer=9±1; n=4), or CD8-depleted LTα KO recipients (titer=13±3; n=3; p<0.001 for all groups compared to WT recipients, signified by “***”). The dotted line represents naïve serum control. C) CD8-mediated in vivo allocytotoxicity was readily detected in WT (87±4%; n=7), splenectomized WT (77±7%; n=4), LTα KO (53±6%; n=3), and splenectomized LTα KO recipients (34±5%; n=3) as compared to naïve controls (2±0.1%; p<0.01 for all, signified by “*”). In vivo cytotoxicity was significantly reduced but still detectable in CD8-depleted WT recipients (45±12%; n=3; p=0.04 compared to WT, signified by “**”). Cytotoxicity was only abrogated in WT recipients following both CD4- and CD8-depletion (0±0%; p<0.001). In contrast, CD8-depletion abrogated in vivo cytotoxicity in LTα KO recipients (5±2%; n=3; p=0.004, signified by “***”).

Figure 4. Hepatocyte transplant directly to the liver (intraportal injection) elicits CD8-dependent rejection (but not alloantibody production) in the absence of CD4⁺ T cells and peripheral lymphoid tissue

FVB/N hepatocytes (H-2^q) were transplanted to the liver by intraportal injection into CD4-deficient wild-type (WT), LTα KO and/or splenectomized (splx) hosts. A) Rejection kinetics were unchanged in the absence of host CD4⁺ T cells in WT, LTα KO and/or splenectomized hosts. Allogeneic hepatocytes were rejected with similar rapid kinetics in CD4-depleted (wild type) hosts (MST=10 days), CD4-depleted hosts with splenectomy (MST=10 days), or CD4-depleted LTα KO hosts with splenectomy (in the absence of both host lymph nodes and spleen) (MST=10 days; p=ns; n=5–6 samples per condition). Following CD8-depletion, splenectomized CD4-deficient WT (n=4) and LTα KO recipients (n=3) exhibit long-term survival of transplanted hepatocytes (MST>30 days; p=0.002). B) CD8-mediated in vivo allocytotoxicity was detected in CD4-depleted WT (68±6%; n=4), CD4-depleted splenectomized WT (43±2%; n=3), CD4-depleted LTα KO recipients (32±4%; n=5), and CD4-depleted splenectomized LTα KO recipients (22±3%; n=4; p<0.005 for all groups compared to naïve control, signified by “*”). Following CD8-depletion, allocytotoxicity was abrogated in CD4-depleted WT (0±0%; n=4) and CD4-depleted LTα KO recipients (4±2%; n=3; p<0.008, signified by “**” compared to CD4-deficient recipients).

Figure 5. Dominant humoral alloimmunity elicited by kidney subcapsular hepatocyte transplantation is critically dependent on CD4⁺ T cells and peripheral lymphoid tissue

FVB/N hepatocytes (H-2^q) were transplanted to the kidney subcapsular site in untreated wild-type (WT) hosts, WT hosts with previous splenectomy, untreated lymph node deficient hosts (LTα KO), and LTα KO hosts with previous splenectomy (all H-2^b). A) Alloantibody production was evaluated in recipient mice on day 14 following transplantation. Following kidney subcapsular transplant, WT recipients (titer=270±60; n=5) and splenectomized WT recipients (titer=210±40; n=3) produced significant amounts of alloantibody. Significantly higher alloantibody levels were observed following CD8-depletion in WT recipients (titer=3300±600, n=3, p<0.001 as compared to WT recipients, as signified by “*”). Alloantibody levels were minimal in LTα KO (titer=10±1; n=3), splenectomized LTα KO recipients (titer=13±3; n=3), and CD8-depleted LTα KO recipients (titer=13±3; n=3; p<0.02 for all comparisons, as signified by “**”). The dotted line represents naïve serum control. B) Rejection of allogeneic hepatocytes after transplant to the kidney subcapsular site was not delayed in the absence of host spleen alone (WT splx, MST=10 days compared to MST=14 days for WT; p=ns). Rejection was significantly delayed in the absence of host lymph nodes (LTα KO, MST=28 days; p=0.001 relative to WT), in the absence of both host lymph nodes and spleen (LTα KO splx MST=49 days; p=0.002 relative to WT; p=ns relative to LTα KO; n=5–6 samples per condition), and in CD8-depleted LTα KO recipients (long-term survival, MST>50 days, n=3, p<0.001). C) In vivo allocytotoxicity was detected in WT (35±2%; n=3), splenectomized WT (36±5%; n=3), and CD8-depleted WT kidney subcapsular recipients (59±3%; n=3; p<0.002 compared to naïve control). In vivo cytotoxicity was negligible in all LTα KO recipients (6±1%; n=3), including splenectomized (5±1%; n=3) or CD8-depleted LTα KO recipients (4±2%; n=3; p=ns for all compared to naïve control).